Live cell imaging by multifocal multiphoton microscopy

Straub, Martin; Lodemann, Peter; Holroyd, P.; Jahn, Reinhard; Hell, Stefan W.

doi:10.1078/0171-9335-00105

Cited by 75 publications

(43 citation statements)

References 35 publications

(42 reference statements)

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“…This instrument bears a resemblance to a number of multifocal multiphoton configurations, whose major advantage is that they increase the image acquisition rate over that of point-scanning systems (12,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Most use widefield detection for parallelized detection of the multiple focal spots and this is important for 2P-MSIM for two critical reasons.…”

Section: Msim Excitation Was Originally Implemented With a Digital MImentioning

confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

109

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Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 μm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.multiphoton | superresolution F luorescence microscopy is an invaluable tool for biologists.Protein distributions in cells have an interesting structure down to the nanometer scale, but features smaller than 200-300 nm are blurred by diffraction in widefield and confocal fluorescence microscopes. Superresolution techniques like photoactivated localization microscopy (1), stochastic optical reconstruction microscopy (2), or stimulated emission depletion (STED) (3) microscopy allow the imaging of details beyond the limit imposed by diffraction, but usually trade acquisition speed or straightforward sample preparation. And although STED can provide resolution down to 40 nm, STED-specific fluorophores are recommended and it often requires light intensities that are orders of magnitude above widefield and confocal microscopy. On the other hand, structured illumination microscopy (SIM) (4) gives twice the resolution of a conventional fluorescence microscope with light intensities on the order of widefield microscopes and can be used with most common fluorophores. SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5, 6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 μm and at ∼1 Hz imaging frequency. In MSIM, images are excited with a multifocal excitation pattern, and the resulting fluorescence in the multiple foci are pinholed, locally scaled, and summed to generate an image [multifocal-excited, pinholed, scaled, and summed (MPSS)] with root 2-improved resolution relative to widefield microscopy, and improved sectioning compared with SIM due to confocal-like pinholing. Deconvolution is applied to recover the final MSIM image which has a full factor of 2 resolution improvement over the diffraction limit.

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Section: Msim Excitation Was Originally Implemented With a Digital MImentioning

confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

109

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“…Fluorescence microscopy has been a powerful technique in cell biology 1,2 following the development of various fluorescent probes 2,3 and the achievement of threedimensional sectioning capability with confocal detection 4 and multiphoton excitation. [5][6][7] However, this technique has two disadvantages, namely, the photobleaching of fluorescent probes and their perturbation of cell functions. 8 Imaging based on the inherent vibrational properties of molecules provides a direct way of chemically mapping an unstained sample.…”

Section: Introductionmentioning

confidence: 99%

Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy

2002

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We present a systematic characterization of coherent anti-Stokes Raman scattering (CARS) microscopy. CARS signal generation in a heterogeneous sample under a tight-focusing condition is formulated by the Green's function method. The CARS radiation pattern and the forward-and backward-detected CARS signals from a three-dimensional Raman scatterer are calculated. The coherent nature of CARS image formation and its consequences for image contrast and spatial resolution are investigated. Experimental implementations of CARS microscopy with collinearly copropagating and counterpropagating excitation beams, forward and backward data collection, and polarization-sensitive detection are described. Finally, CARS images of unstained live cells with forward detection, epidetection, and polarization-sensitive detection are presented and compared.

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“…Live cell imaging, for instance, requires fast image acquisition such that the cells and intracellular components do not change significantly during acquisition [8]. Furthermore, in high content screening (HCS) a technique used in drug discovery, it requires a large number of images to be collected over a wide range of experimental conditions in order to establish a suitable set for statistical analysis [9].…”

Section: Introductionmentioning

confidence: 99%

High-speed multifocal array scanning using refractive window tilting

Tsikouras

Berman²,

Andrews

et al. 2015

Biomed. Opt. Express

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Confocal microscopy has several advantages over wide-field microscopy, such as out-of-focus light suppression, 3D sectioning, and compatibility with specialized detectors. While wide-field microscopy is a faster approach, multiplexed confocal schemes can be used to make confocal microscopy more suitable for high-throughput applications, such as high-content screening (HCS) commonly used in drug discovery. An increasingly powerful modality in HCS is fluorescence lifetime imaging microscopy (FLIM), which can be used to measure protein-protein interactions through Förster resonant energy transfer (FRET). FLIM-FRET for HCS combines the requirements of high throughput, high resolution and specialized time-resolving detectors, making it difficult to implement using wide-field and spinning disk confocal approaches. We developed a novel foci array scan method that can achieve uniform multiplex confocal acquisition using stationary lenslet arrays for high resolution and high throughput FLIM. Unlike traditional mirror galvanometers, which work in Fourier space between scan lenses, this scan method uses optical flats to steer a 2-dimension foci array through refraction. After integrating this scanning scheme in a multiplexing confocal FLIM system, we demonstrate it offers clear benefits over traditional mirror galvanometer scanners in scan linearity, uniformity, cost and complexity. 277-296 (2015). 27. T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J.

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Live cell imaging by multifocal multiphoton microscopy

Cited by 75 publications

References 35 publications

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy

High-speed multifocal array scanning using refractive window tilting

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